Compound libraries by glycotransferases and a method of preparing the same

ABSTRACT

The present invention aims to provide processes for rapidly and easily preparing compound libraries of known structures, and compound libraries prepared by said processes. A compound library of the present invention is prepared by a process for preparing a compound library in the same vessel, comprising: (1) mixing one or more donor substrates, acceptor substrates, and transferases; (2) performing a transfer reaction to reach a degree of transfer of 1%-99% by incubating the mixture; and (3) stopping the transfer reaction.

FILED OF THE INVENTION

The present invention relates to compound libraries rapidly and easilyprepared in the same vessel using transferases and processes forpreparing them.

BACKGROUND OF THE INVENTION

In understanding sophisticated regulatory mechanisms of organisms, it isvery important to investigate interactions between biological moleculesand correlations between the structures and functions of biologicalmolecules. Isolating nucleic acids or proteins expressed in only smallamounts in tissues or cells of organisms and rapidly and convenientlyidentifying their sequence information or three-dimensional structuresis an important challenge in the field of bioinformatics aiming topresume molecular functions from molecular structures or in the field ofproteomics aiming to exhaustively analyze expressed proteins.

Biological molecules that control sophisticated regulatory mechanismsinclude glycoconjugates or the like in addition to nucleic acids,proteins, and peptides. For example, functions of proteins translatedafter gene expression in cells are regulated by variouspost-translational modifications such as activation by various proteasesor activity regulation via glycosylation, sulfation, phosphorylation,acylation or the like by various transferases. It is also known thatabout 50% or more of biological proteins exist as complexes witholigosaccharides and that oligosaccharides play the role of controllingthe structures and functions of glycoproteins. Moreover, the functionsof oligosaccharides per se are also controlled by sulfation,phosphorylation, acylation, or addition of new glycosyl residues, asdescribed above.

However, when one wishes to analyze the functions of modified proteins,peptides, oligosaccharides or the like, multiple modifications arefrequently seen in the same molecule rather than uniform modification ineach molecule. Thus, such non-uniformity of modified molecules makes itnot only very difficult to closely analyze molecular interactions orcorrelations between the structures and functions of molecules, but alsodifficult to simultaneously obtain a plurality of target modifiedmolecules in such studies.

At present, many physiologically active peptides are known. It is veryimportant to investigate relationships between the amino acid sequencesof such peptides and their physiological activity, but there are fewexamples of studies that went into details of physiological activityusing post-translationally modified peptides observed in vivo. If alibrary of various modified peptides could be available in such studies,it would become possible not only to easily analyze the relationshipbetween the amino acid sequence and physiological activity of a specificpeptide by closely examining the physiological activity of the peptideobtained by screening, but also to search modified peptides havingdesired activity.

The same problem occurs in the field of study of glycoproteins andoligosaccharides, because oligosaccharide moieties also undergo variousmodifications, i.e. a wide variety of glycosyl residues are added to anacceptor substrate at specific times and specific locations by theaction of multiple enzymes expressed in vivo, including enzymesresponsible for oligosaccharide elongation such as glycosyltransferases,glycosidases and glycosylation enzymes or transferases required forsulfation, phosphorylation, acylation or the like as described above. Itcan be said that such modifications to oligosaccharides are importantmechanisms as seen with protein phosphorylation cascades.

For example, oligosaccharides added to proteins are classified broadlyinto O-linked oligosaccharides attached to serine/threonine residues andN-linked oligosaccharides attached to asparagine residues of proteins orpeptides, and exist as variations of oligosaccharide structuresdepending on the type, number and mode of linkage of glycosyl residuesconstituting the oligosaccharides. Such oligosaccharide structures arestrictly controlled by the glycosyltransferases or the like describedabove during the expression of glycoproteins. Sulfation ofoligosaccharides, which means the sulfotransferase-catalyzed transfer ofa sulfate group from a sulfate donor, 3′-phosphoadenosine5′-phosphosulfate (PAPS) to an acceptor substrate, has become known tobe responsible for various biological phenomena. In this manner, thereis a high possibility that the structural analysis of modifiedoligosaccharides or those oligosaccharides per se may be applied tobiotechnology or medical field. However, it is difficult to obtainvarious oligosaccharides or oligosaccharide libraries for use in thestudy of oligosaccharides including analysis of the importance ofmodification of oligosaccharides because oligosaccharides are expressedin vivo in very small amounts.

Known methods for supplying oligosaccharides or oligosaccharidelibraries include combinatorial chemical synthesis based on solid-phasesynthesis (see Non-patent documents 1-4) and combinatorial chemicalsynthesis based on liquid-phase synthesis (see Non-patent documents5-9).

Examples of combinatorial chemical syntheses based on solid or liquidphase include Split and Combine Library Synthesis, Parallel Synthesis ofCompound arrays, and One-Pot Glycosylation as technically distinctapproaches. However, these methods mainly use organic chemical synthesisand therefore inevitably require the step of protection/deprotection ineach synthetic process because a suitable protective group must beintroduced in advance into the hydroxyl group of the glycosyl donorsubstrate or glycosyl acceptor substrate to newly add a glycosyl residueto a specific site of an existing glycosyl residue. If an unprotectedacceptor substrate was used (e.g., random glycosylation), the resultingproduct would include a lot of mixtures of steric isomers and positionisomers and purification or structure determination thereof would becomplex.

Conventional enzymatic or chemical-enzymatic synthesis uses a glycosylacceptor substrate mainly prepared by chemical synthesis to performglycosylation using glycosidase-catalyzed reverse reaction orglycosyltransferase-catalyzed sequential synthesis. For example, a basicprocess for synthesizing an oligosaccharide containing several glycosylresidues comprises mixing a glycosyl acceptor substrate, a glycosyldonor substrate, and a glycosyltransferase, recovering/purifying theproduct after the glycosyl transfer to the substrate has proceeded to100%, and performing oligosaccharide elongation on said product by atransfer reaction using a different (or the same) glycosyltransferase asa glycosyl acceptor substrate for the subsequent reaction. However, suchenzymatic synthesis requires complex steps including many reactions,recovery, and purification to obtain a desired oligosaccharide structureand also requires that separately prepared oligosaccharides should begathered to construct an oligosaccharide library. Moreover, theoligosaccharide library contains very limited kinds of oligosaccharidestructures because of the limitation of the kinds ofglycosyltransferases commercially or otherwise available.

A known method for preparing an oligosaccharide library by cellularsynthesis rather than organic synthesis is biocombinatorial synthesis(see Non-patent document 10). This method uses oligosaccharide primersmimicking the oligosaccharide structures serving as precursors in theoligosaccharide biosynthetic pathways as artificial substrates forglycosyltransferase reactions. Specifically, oligosaccharide primers areintroduced into a cell so that various oligosaccharides can be obtainedby oligosaccharide elongation using the oligosaccharide biosyntheticpathways of the cell. An oligosaccharide library is constructed bychanging the combination of the oligosaccharide primers and cell usedbecause the biosynthetic pathways vary with the cell type. However,various oligosaccharide-related enzymes such as glycosyltransferases areexpressed in cells at different expression times and levels even if cellculture conditions slightly changes. Moreover, it is not only difficultto construct variations of oligosaccharide structures at will but alsocomplex to purify the products or separate isomers and determine theirstructures because oligosaccharide elongation depends on thebiosynthetic pathways of each cell. Thus, it would be desirable tostably supply oligosaccharide libraries applicable to biotechnology ormedical field.

On the other hand, our research group already identified many kinds ofglycosyltransferases and sulfotransferases that were difficult to obtaindespite their indispensability for oligosaccharide elongation oroligosaccharide modification and explained their gene structures.Specifically, our research group identified ppGalNAc-T10 (see Non-patentdocument 11), T12 (see Non-patent document 12), T13 (see Non-patentdocument 13), T14 (see Non-patent document 14), T15 (see Non-patentdocument 15), and T16, T17, and T18 (see Patent document 1) astransferases of N-acetylgalactosamine (GalNAc) having the activity oftransferring N-acetylgalactosamine to the hydroxyl group of serine orthreonine residues of core proteins or peptide sequences via al linkage.It should be noted that ppGalNAc-T16, T17, and T18 mentioned abovecorrespond to GalNAc-T11, T16, and T15 in Patent document 1,respectively. Our research group also identifiedN-acetylgalactosaminyltransferases transferring N-acetylgalactosamine toacceptor substrates other than those described above, e.g., glucuronicacid or N-acetylglucosamine (see Non-patent documents 16-22).

Our research group also identified β3GalT5 as a galactosyltransferasehaving the activity of transferring galactose to N-acetylglucosamine(see Non-patent document 23). We also identified β3GnT2, T3, and T4 (seeNon-patent document 24), and β3GnT5 (see Non-patent document 25) asN-acetylglucosaminyltransferases having the activity of transferringN-acetylglucosamine to galactose. We also identified heparan sulfate3-O-sulfotransferase (see Non-patent document 26) as a sulfotransferase.

Thus, oligosaccharide libraries having specific structures can beconstructed by taking advantage of benefits from ensuring a stablesupply of many kinds of glycosyltransferases and sulfotransferases asdescribed above and defining the substrate specificity of eachtransferase.

Moreover, such transferases can be applied to not only oligosaccharideelongation or oligosaccharide modification but also glycosylation ofvarious compounds such as proteins, peptides and lipids as well asconstruction of compound libraries having sulfated, phosphorylated oracylated structures. Construction of compound libraries of the presentinvention can also be expected to give a clue to the explanation ofmodification mechanisms after gene expression in vivo by analyzing thesubstrate specificity of transferases or the like in vitro.

SUMMARY OF THE INVENTION

An object of the present invention is to provide novel processes forrapidly and easily preparing compound libraries of known structures.Another object of the present invention is to provide compound librariesprepared by said processes and uses thereof.

Our research group accomplished the present invention on the basis ofthe finding that compound libraries having multiple components of knownstructures can be rapidly and easily prepared by using transferreactions based on substrate specificity and further performing the sameor different transfer reaction before the previous transfer reaction hasbeen completely terminated. Moreover, it was found that the compositionratios or other analyses of components contained in the compoundlibraries can be conveniently determined.

Accordingly, the present invention provides a process for preparing acompound library in the same vessel, comprising: (1) mixing one or moredonor substrates, acceptor substrates, and transferases; (2) performinga transfer reaction to reach a degree of transfer of 1%-99% byincubating the mixture; and (3) stopping the transfer reaction.

Technical features of the process of the present invention are explainedby way of an embodiment of a process for preparing an oligosaccharidelibrary using a glycosyltransferase as a transferase, a glycosyl donorsubstrate as a donor substrate, and a glycosyl acceptor substrate as anacceptor substrate. For example, when a glycosyl acceptor substrateβ4Gal-core 2 is used as a starting material and various glycosyl donorsubstrates and glycosyltransferases are added to sequentially performtransfer reactions as shown in scheme 1, an oligosaccharide libraryhaving eight different oligosaccharide structures can be constructedfrom a total of four glycosyl transfer reactions by allowing a part ofthe glycosyl acceptor substrate to remain unreacted in each reactionstep.

Scheme 1 is explained more specifically. When the starting materialβ4Gal-core 2 is designated sugar A and glycosylated oligosaccharidesafter reactions are designated sugar B, sugar C, etc., the reaction ofthe first stage in scheme 1 involves mixing UDP-N-acetylneuraminic acid(open star) (donor substrate), sugar A (acceptor substrate), and anN-acetylneuraminyltransferase (ST3Gal IV) (transferase) and performing atransfer reaction at a predetermined temperature for a predeterminedperiod. In this case, the starting material sugar A and sugar B withN-acetylneuraminic acid attached are produced by keeping the degree ofglycosylation (reaction) below 100%. Then, the reaction of the secondstage involves mixing sugar A and sugar B (reaction products) withUDP-N-acetylglucosamine (solid square) (donor substrate) and anN-acetylglucosaminyltransferase (β3GnT2) (transferase) and reacting themixture at a predetermined temperature for a predetermined period togive a compound library having three different oligosaccharidestructures consisting of sugar A, sugar C, and sugar B based onsubstrate specificity by keeping the degree of glycosylation below 100%in the same manner as in the reaction of the first stage. Eightoligosaccharide structures can be obtained from one starting material(sugar A) by repeating similar reactions up to the fourth stage. Forcomparison, conventional enzymatic synthesis affords only theoligosaccharide composed of the starting material (sugar A) withN-acetylneuraminic acid (open star) and fucose (open triangle) attachedas shown at the right end on the bottom line in scheme 1 even ifreactions were similarly carried out until the fourth stage in theexample shown in scheme 1 because conventional methods are essentiallyintended to reach the degree of glycosylation of 100%. Thus, anoligosaccharide library having multiple oligosaccharide structures canbe rapidly and easily obtained when an oligosaccharide compound is usedas a starting material along with a glycosyltransferase and a glycosyldonor substrate, for example. Moreover, it will be understood thatcompound libraries containing multiple structures can be prepared byusing other starting materials, transferases and donor substrateswithout being limited to oligosaccharides, glycosyltransferases andglycosyl donor substrates so that the present invention provides abeneficial method for supplying such compound libraries.

In one embodiment of the present invention, the process may furthercomprise the step of repeating steps (1)-(3) using the same or differentdonor substrate and transferase as or from the donor substrate andtransferase used in step (1). In this case, the product obtained afterstopping the transfer reaction in step (3) serves as the acceptorsubstrate in step (1) of the subsequent cycle. Any number of cycles ofsteps (1)-(3) can be repeated until a compound library containing adesired structure, and the number of cycles can be determined by thoseskilled in the art depending on the purpose. The number of cycles ispreferably one or more, more preferably 1-50, still more preferably1-10, further more preferably 1-8, most preferably 1-6.

In one embodiment of the present invention, the degree of transferreaction by the transferase may not reach 100% in the process forpreparing a compound library of the present invention. That is, it israther preferred that a part of the acceptor substrate remains unreactedby stopping the reaction before the transfer reaction proceeds to 100%in order to prepare a compound library containing components havingdifferent structures. The transfer reaction is preferably stopped whenthe degree of transfer reaches about 1%-about 99%, more preferably about5%-about 95%, still more preferably about 10%-about 90%, further morepreferably about 20%-about 80%, still further more preferably about30%-about 70%, even more preferably about 40%-about 60%, most preferablyabout 50%. It will be readily appreciated that the compound librarycontains the most uniform amounts of components when the transferreaction is stopped at the degree of reaction of 50% in step (3).

In one embodiment of the present invention, a mixture at a mid stage forconstructing a library can be used as a starting material forconstructing another library in the process for preparing a compoundlibrary of the present invention.

In one embodiment of the present invention, the order of transferasesused in the process for preparing a compound library of the presentinvention is not limited, but those skilled in the art can select thetypes of transferases and the order of adding them in consideration ofthe substrate specificity of the transferases to prepare a compoundlibrary having a specific structure. On the other hand, compoundlibraries not containing a specific structure can also be prepared basedon the substrate specificity of transferases.

In one embodiment of the present invention, transferases used in theprocess for preparing a compound library of the present inventioninclude, but not limited to, N-acetylneuraminyltransferases,fucosyltransferases, N-acetylglucosaminyltransferases,N-acetylgalactosaminyltransferases, galactoseyltransferases,glucosyltransferases, glucuronyltransferases, mannosyltransferases,xylosyltransferases, sulfotransferases, phosphotransferases, andacyltransferases.

In one embodiment of the present invention, the acceptor substrate usedin the process for preparing a compound library of the present inventionis not specifically limited so far as transferases can transfer asubstituent such as a glycosyl residue, sulfate group, phosphate groupor acyl group to it from a donor substrate on the basis of the substratespecificity. Preferably, it is a glycosyl acceptor substrate, protein,peptide, amino acid or lipid or a modified form thereof, more preferablya glycosyl acceptor substrate or peptide, still more preferably aglycosyl acceptor substrate. The glycosyl acceptor substrate ispreferably a glycopeptide, glycoprotein, monosaccharide,oligosaccharide, glycolipid, protein or peptide or a modified formthereof, more preferably a glycopeptide, glycoprotein or oligosaccharideor a modified form thereof, still more preferably a glycopeptide oroligosaccharide or a modified form thereof.

In one embodiment of the present invention, donor substrates used in theprocess for preparing a compound library of the present inventioninclude, but not limited to, sugar nucleotides, dolicholphosphate-sugars, 3′-phosphoadenosine 5′-phosphosulfate (PAPS),adenosine triphosphate (ATP), and acetyl-CoA.

According to the present invention, a compound library prepared by theprocess for preparing a compound library of the present invention isprovided.

In one embodiment of the present invention, a compound library isprovided wherein the structure of each component of the compound libraryis identified from its molecular weight. Methods for determining thestructure of each component from its molecular weight include massspectrometry, electrophoresis, and gel filtration.

According to the present invention, a chip using the compound library ofthe present invention is provided. Compounds used in the chip areobtained by the process for preparing a compound library of the presentinvention. In one embodiment of the present invention, each componentcontained in the compound library can be used afterisolation/purification. In one embodiment of the present invention, eachcomponent contained in the compound library can also be used withoutisolation/purification.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows the results of measurements by using Reflex IV (massspectrometer) for the oligosaccharide structures obtained by fourtransfer reactions from a glycopeptide having β4Gal-core 2 structure asa starting material (glycosyl acceptor substrate) with variousglycosyltransferases and sugars (glycosyl donor substrates). Sixoligosaccharides were obtained by the end of reaction 4. In the figure,solid circles represent N-acetylgalactosamine, open circles representgalactose, solid squares represent N-acetylglucosamine, open trianglesrepresent fucose, and open stars represent N-acetylneuraminic acid.

FIG. 2 shows the results of measurements by using Reflex IV (massspectrometer) for the oligosaccharide structures obtained by fourtransfer reactions from a glycopeptide having β4Gal-core 2 structure asa starting material (glycosyl acceptor substrate) with variousglycosyltransferases and sugars (glycosyl donor substrates). The orderof reaction 2 and reaction 3 in FIG. 1 was changed. Eightoligosaccharides were obtained by the end of reaction 4′. In the figure,solid circles represent N-acetylgalactosamine, open circles representgalactose, solid squares represent N-acetylglucosamine, open trianglesrepresent fucose, and open stars represent N-acetylneuraminic acid.

FIG. 3 shows the results of measurements by using Reflex IV (massspectrometer) for the lactosamine structure (Galβ1-4GlcNAcβ1-3) obtainedin one reaction from Tn-Muc1a as a starting material with two glycosyldonor substrates (UDP-GlcNAc and UDP-Gal) and three glycosyltransferases(two N-acetylglucosaminyltransferases and a galactosyltransferase). Inthe figure, solid circles represent N-acetylgalactosamine, solid squaresrepresent N-acetylglucosamine, and open circles represent galactose.

FIG. 4 shows the results of measurements by using Reflex IV (massspectrometer) for the oligosaccharide structures obtained by using core3-Muc1a structure as a starting material with anN-acetylglucosaminyltransferase (β3GnT2), a galactosyltransferase(β4GalT1), a glycosyl acceptor substrate (core 3-Muc1a), and glycosyldonor substrates (UDP-GlcNAc and UDP-Gal) to prepare oligosaccharideshaving a polylactosamine structure (reaction A), which were furtherreacted with a sialyltransferase (ST3GalIII) and a glycosyl donorsubstrate (CMP-Neu5Ac) to prepare polylactosamine chains sialylated atthe non-reducing end. In the figure, solid circles representN-acetylgalactosamine, solid squares represent N-acetylglucosamine, opencircles represent galactose, and open stars represent N-acetylneuraminicacid.

DETAILED DESCRIPTION OF THE INVENTION

For explaining the present invention, preferred embodiments aredescribed in detail below.

1. Transferases

(1) Glycosyltransferases

Glycosyltransferases are proteins that catalyze the transfer of aglycosyl residue from a glycosyl donor substrate (e.g., sugarnucleotides) to a glycosyl acceptor substrate (e.g., glycopeptides,peptides). The catalytic reaction is expressed by the reaction formula:glycosyl acceptor substrate+sugar 1-nucleotide

sugar 1-glycosyl acceptor substrate+nucleotide  formula (A).

In formula (A), the product with sugar 1 attached “sugar 1-glycosylacceptor substrate” serves as the glycosyl acceptor substrate in thesubsequent reaction. For example, if the glycosyl acceptor substrate isreacted with sugar 2-nucleotide in the presence of a glycosyltransferasetransferring sugar 2, the product is sugar 2-sugar 1-glycosyl acceptorsubstrate when the reaction proceeds to 100%.

As described above, our research group already succeeded in cloninggenes encoding N-acetylgalactosaminyltransferases,galactosyltransferases, N-acetylglucosaminyltransferases,fucosyltransferases, glucuronyltransferases and sulfotransferases havingvarious substrate specificities.

(a) N-acetylgalactosaminyltransferases

N-acetylgalactosaminyltransferases are proteins that catalyze thetransfer of an N-acetylgalactosamine residue to a glycosyl acceptorsubstrate. Eighteen human N-acetylgalactosaminyltransferasestransferring N-acetylgalactosamine to the hydroxyl group of anon-glycosylated acceptor substrate serine/threonine have been known sofar. Our research group already isolated genes encoding pp-GalNAc-T10,T12, T14, T15, T16, T17, and T18, and determined their nucleotidesequences and putative amino acid sequences (see Patent document 1). Thenucleotide sequences, putative amino acid sequences, substratespecificities, and expression distributions in tissues of the nucleicacids encoding these N-acetylgalactosaminyltransferases are disclosed inPatent document 1. It should be noted that pp-GalNAc-T10 corresponds toGalNAc-T13 disclosed in Patent document 1, pp-GalNAc-T12 corresponds toGalNAc-T14, pp-GalNAc-T14 corresponds to GalNAc-T12, pp-GalNAc-T15corresponds to GalNAc-T17, pp-GalNAc-T16 corresponds to GalNAc-T11,pp-GalNAc-T17 corresponds to GalNAc-T16, and pp-GalNAc-T18 correspondsto GalNAc-T15, respectively (see Table 1). All of the otherN-acetylgalactosaminyltransferases are disclosed in prior technicaldocuments described in Table 1.

Our research group also identified glycosyltransferases belonging toN-acetylgalactosaminyltransferases but transferringN-acetylgalactosamine to glycosyl acceptor substrates other than thosedescribed above, e.g., glucuronic acid or N-acetylglucosamine (seeNon-patent documents 16-22).

Although the N-acetylgalactosaminyltransferases described above arespecific for glycosyl acceptor substrates having serine/threonine,glucuronic acid, or N-acetylglucosamine,N-acetylgalactosaminyltransferases used in processes for preparing anoligosaccharide library of the present invention are not specificallylimited so far as they transfer N-acetylgalactosamine. TABLE 1N-acetylgalactosaminyltransferase and substrate specificity thereof Namein International Substrate Publication Name Origin specificityReferences W003/057887 pp-GalNAc-T1 Human Ser/Thr White, T. et al.(1995) pp-GalNAc-T2 Human Ser/Thr White, T. et al. (1995) pp-GalNAc-T3Human Ser/Thr Bennet, E. P. et al. (1996) pp-GalNAc-T4 Human Ser/ThrBennet, E. P. et al. (1998) pp-GalNAc-T5 Rat Ser/Thr Ten Hagen, K. G. etal. (1998) pp-GalNAc-T6 Human Ser/Thr Bennet, E. P. et al. (1999) (1)pp-GalNAc-T7 Human Ser/Thr Bennet, E. P. et al. (1999) (2) pp-GalNAc-T8Human Ser/Thr White, K. E. et al. (2000) pp-GalNAc-T9 Human Ser/ThrToba, S. et al. (2000) pp-GalNAc-T10 Rat Ser/Thr Ten Hagen, K. G. et al.(2001) pp-GalNAc-T10 Human Ser/Thr Cheng, L. et al. (2002) GalNAc-T13pp-GalNAc-T11 Human Ser/Thr Schwientek, Y. et al. (2002) pp-GalNAc-T12Human Ser/Thr Guo, J. M. et al. (2002) GalNAc-T14 pp-GalNAc-T13 HumanSer/Thr Zhang, Y. et al. (2003) pp-GalNAc-T14 Human Ser/Thr Wang, H. etal. (2003) GalNAc-T12 pp-GalNAc-T15 Human Ser/Thr Cheng, L. et al.(2004) GalNAc-T17 pp-GalNAc-T16 Human Ser/Thr International PublicationGalNAc-T11 Number: W003/057887 pp-GalNAc-T17 Human Ser/Thr InternationalPublication GalNAc-T16 Number: W003/057887 pp-GalNAc-T18 Human Ser/ThrInternational Publication GalNAc-T15 Number: W003/057887

(b) Galactosyltransferases

Our research group already identified β3GalT5 as a galactosyltransferasethat catalyzes the transfer of galactose to N-acetylglucosamine (seeNon-patent document 23). Although these galactosyltransferases arespecific for glycosyl acceptor substrates having N-acetylglucosamine,galactosyltransferases used in processes for preparing anoligosaccharide library of the present invention are not specificallylimited so far as they transfer galactose.

(c) N-acetylglucosaminyltransferases

Our research group already identified β3GnT2, T3, T4 (see Non-patentdocument 24), and P3GnT5 (see Non-patent document 25) asN-acetylglucosaminyltransferases that catalyze the transfer ofN-acetylglucosamine to galactose. Although theseN-acetylglucosaminyltransferases are specific for acceptor substrateshaving N-acetylglucosamine, N-acetylglucosaminyltransferases used inprocesses for preparing a compound library of the present invention arenot specifically limited so far as they transfer N-acetylglucosamine.

(d) Other Glycosyltransferases

Glycosyltransferases used in processes for preparing a compound libraryof the present invention other than those described above include, butnot limited to, N-acetylneuraminyltransferases, fucosyltransferases,glucuronyltransferases, glucosyltransferases, mannosyltransferases, andxylosyltransferases. Some of these glycosyltransferases are commerciallyor otherwise available.

Generally, it is well known that proteins having a physiologicalactivity such as glycosyltransferases may retain the physiologicalactivity even when one or more amino acids in their amino acid sequencesare substituted or deleted or one or more amino acids are inserted oradded into the amino acid sequences. It is also known that naturallyoccurring proteins include variant proteins having one or more aminoacid changes resulting from the genetic variation between varieties ofspecies producing them or between ecotypes or the presence of verysimilar isozymes. Therefore, glycosyltransferases having an amino acidsequence obtained by substituting or deleting one or more amino acids inan amino acid sequence already known or by inserting or adding one ormore amino acids into such an amino acid sequence are also included inthe scope of the present invention so far as they have the transferaseactivity described above.

(2) Other Transferases

Transferases used in processes for preparing a compound library of thepresent invention other than the glycosyltransferases described aboveinclude any transferases capable of transferring a substituent from adonor substrate to an acceptor substrate without limitation. Suchtransferases include sulfotransferases, phosphotransferases, andacyltransferases.

(a) Sulfotransferases

Sulfotransferases catalyze the reaction transferring sulfate from asulfate donor substrate active sulfate (3′-phosphoadenosine5′-phosphosulfate: PAPS) to the hydroxyl group, amino group, or thiolgroup of an acceptor substrate. In post-translational modification ofproteins, sulfate is introduced into the hydroxyl group on the sidechain of an amino acid residue tyrosine to control the activity of theproteins. Sulfation in oligosaccharides varies especially inglycosaminoglycans. Sulfated glycosaminoglycans have a repeatingdisaccharide structure and include chondroitin sulfate, heparan sulfate,dermatan sulfate, and keratan sulfate. For example, chondroitin sulfateis basically composed of a glucuronic acid residue and anN-acetylgalactosamine residue sulfated at different sites depending onthe type of sulfotransferase.

Sulfotransferases used in processes for preparing a compound library ofthe present invention are not specifically limited so far as theytransfer sulfate. Preferred are tyrosine sulfotransferases, chondroitinsulfotransferases, heparan sulfate N-sulfotransferases, heparan sulfateO-sulfotransferases, keratan sulfotransferases, dermatan/chondroitinsulfate 2-sulfotransferases, β-galactose 3-O-sulfotransferases, NHK-1sulfotransferases, galactose/N-acetylgalactosamine/N-acetylglucosamine6-O-sulfotransferases (GSTs), and N-acetylgalactosamine4-O-sulfotransferases, more preferably β-galactose3-O-sulfotransferases, NHK-1 sulfotransferases, N-acetylglucosamine6-O-sulfotransferases, and N-acetylgalactosamine 4-O-sulfotransferases.Sulfotransferases are described in, e.g., Handbook ofGlycosyltransferase and Related Genes.

(b) Phosphotransferases

Phosphotransferases (kinases) catalyze the reaction transferringphosphate from a phosphate donor (e.g., adenosine 5′-triphosphate) tothe hydroxyl group of an acceptor substrate. In post-translationalmodification of proteins, phosphate is introduced into the hydroxylgroup on the side chain of an amino acid residue tyrosine, serine orthreonine to control the activity of the proteins. Phosphorylation inoligosaccharides is predominant in various metabolism pathways. Forexample, hexokinases are involved in the pathway synthesizing glucose6-phosphate from glucose.

Phosphotransferases used in processes for preparing a compound libraryof the present invention are not specifically limited so far as theytransfer phosphate. Preferred are tyrosine kinases, serine-threoninekinases, hexokinases, and N-acetylglucosamine kinases, more preferablyhexokinases, and N-acetylglucosamine kinases. Phosphotransferases aredescribed in, e.g., Non-patent documents 27-29.

(c) Acyltransferases

Acyltransferases catalyze the reaction transferring an acyl group froman acyl donor substrate acetyl-CoA to the carboxyl group, hydroxylgroup, amino group, or thiol group of an acceptor substrate. Especially,acyltransferases are involved in the metabolism of steroids and fattyacids. In post-translational modification of proteins, an acyl group isintroduced into the carboxyl group on the side chain of an amino acidresidue aspartic acid or glutamic acid to control the activity of theproteins. Acylation in oligosaccharides is known to play a pivotal rolefor controlling various biological processes. For example,sialate-4-O-acetyltransferases are involved in the reaction transferringacetyl to the hydroxyl group at the 4-position of a sialic acid attachedto the non-reducing end side.

Acyltransferases used in processes for preparing a compound library ofthe present invention are not specifically limited so far as theytransfer an acyl group. Preferred are sterol O-acyltransferases,sialate-4-O-acetyltransferases, sialate-7-(9)-O-acetyltransferases, andgalactose O-acetyltransferases, more preferablysialate-4-O-acetyltransferases, sialate-7-(9)-O-acetyltransferases, andgalactose O-acetyltransferases. Acyltransferases are described in, e.g.,Non-patent documents 30 and 31.

Any transferases that can be used in processes for preparing a compoundlibrary of the present invention are not limited in their origins orpreparation processes so far as they have characteristics describedabove. That is, transferases may be any of naturally occurring proteins,proteins expressed from recombinant DNAs by genetic engineeringtechniques, or chemically synthesized proteins. The species from whichtransferases are derived are not limited, but preferably animals,microorganisms, and plants, more preferably mammals, E. coli, yeasts,and archaebacteria, still more preferably humans, rats, mice, xenopus,hamsters, and monkeys, further more preferably humans, rats, and mice.

Transferase used in processes for preparing a compound library of thepresent invention also include variants of transferases altered bygenetic engineering techniques to increase or decrease substratespecificity. Construction of such variants can be performed by thoseskilled in the art using well known methods.

2. Acceptor Substrates and Donor Substrates

(1) Acceptor Substrates

Transfer reactions to acceptor substrates such as glycosylation,sulfation, phosphorylation, and acylation are performed by transferringvarious substituents to acceptor substrates in the presence atransferase as a catalyst suitable for the type of substituent. Forexample, it is known that N-acetylgalactosamine residues aresubstrate-specifically transferred to at least the hydroxyl group ofserine/threonine, glucuronic acid, and N-acetylglucosamine as acceptorsubstrates in proteins or peptides when the transferase is anN-acetylgalactosaminyltransferase.

As used herein the term “acceptor substrate” means a compound to which asubstituent is transferred by various transferases on the basis of theirsubstrate specificity. The acceptor substrate is not limited so far asit is a compound having a functional group (e.g., hydroxyl group,carboxyl group, amino group) to which a substituent is transferred by atransferase. The acceptor substrate is preferably a glycosyl acceptorsubstrate, protein, peptide or lipid, more preferably a glycosylacceptor substrate or peptide, still more preferably a glycosyl acceptorsubstrate. The term “glycosyl acceptor substrate” means a substrate towhich a sugar is transferred. Preferably, it is a glycopeptide,glycoprotein, monosaccharide, oligosaccharide, glycolipid, protein orpeptide or a modified form thereof, more preferably a glycopeptide,glycoprotein or oligosaccharide or a modified form thereof, still morepreferably a glycopeptide or oligosaccharide or a modified form thereof.The glycosyl residue constituting the glycosyl acceptor substrate is notlimited to naturally derived sugars, oligosaccharides or the like, butmay also be a modified form thereof. The modified form means aderivative in which one or more hydroxyl groups in the glycosyl residueare replaced by a sulfate group, phosphate group, purine base,pyrimidine base, alkyl group, acyl group, amide group, or amino group.

The moieties other than the sugar moiety of the glycosyl acceptorsubstrate include, but not limited to, proteins, peptides, lipids, alkylgroups, acyl groups, amino groups, hydrazides and oximes that can beattached to any hydroxyl group of the sugar moiety (preferably thehydroxyl group at the reducing end). Thus, the glycosyl acceptorsubstrate used in processes for preparing a compound library of thepresent invention is preferably a protein (or peptide), glycoprotein (orglycopeptide) (e.g., O-glycans, N-glycans), glycolipid, oligosaccharide,monosaccharide (e.g., N-acetylglucosamine, glucuronic acid,N-acetylgalactosamine, galactose, glucose, mannose, fucose) or aminoacid (e.g., serine, threonine, asparagine) or a modified form thereof,more preferably a glycopeptide, glycolipid, oligosaccharide ormonosaccharide or a modified form thereof, still more preferably aglycopeptide or glycolipids or a modified form thereof, most preferablya glycopeptide. The “O-glycan” is a common name of a glycoprotein orglycopeptide consisting of an oligosaccharide linked to theserine/threonine residue of a protein or peptide and is classified byoligosaccharide structure into Tn antigen, core 1, core 2, core 3, core4, core 5, core 6, core 7, and core 8. Here, Tn antigen is GalNAcα1-,core 1 is Galβ1-3GalNAcα1-, core 2 is GlcNAcβ1-6(Galβ1-3)GalNAcα1-, core3 is GlcNAcβ1-3GalNAcα1-, core 4 is GlcNAcβ1-6(GlcNAcβ1-3)GalNAcα1-,core 5 is GalNAcα1-3GalNAcα1-, core 6 is GlcNAcβ1-6GalNAcα1-, core 7 isGalNAcα1-6GalNAcα1-, and core 8 is Galα1-3GalNAcα1-.

(2) Donor Substrates

Transfer reaction means the transfer of a substituent constituting adonor substrate to an acceptor substrate in the presence of atransferase as a catalyst. For example, when a sialic acid residue(e.g., Neu5Ac) is to be transferred to an acceptor substrate havingβ4Gal-core 2 structure, CMP-Neu5Ac can be used as a donor substrate.

As used herein, the term “donor substrate” means a compound constitutinga substituent to be transferred by a transferase on the basis of itssubstrate specificity when the transferase recognizes an acceptorsubstrate. The donor substrate is not specifically limited so far as itdoes not affect substrate specificity of transferases and provides asource for transferring a substituent to an acceptor substrate.Preferably, it is a sugar nucleotide, dolichol phosphate-sugar,3′-phosphoadenosine 5′-phosphosulfate (PAPS), adenosine triphosphate(ATP) or acetyl-CoA, more preferably a sugar nucleotide, dolicholphosphate-sugar or 3′-phosphoadenosine 5′-phosphosulfate (PAPS), mostpreferably a sugar nucleotide.

3. Processes for Preparing a Compound Library

Processes for preparing a compound library of the present inventioninvolve a reaction transferring a substituent of a donor substrate to anacceptor substrate in the same vessel, and are characterized in that adiverse compound library is constructed by sequentially addingtransferases to a mixture of a compound with a substituent attached andan unreacted compound with no substituent attached after stopping thereaction or not before the degree of transfer by each transferasereaches 100%. In processes of the present invention, transfer reactionscan be continuously performed without recovering and purifying theproduct in each reaction step and compound libraries having multiplesubstituents can be constructed, in contrast to conventional enzymaticsynthesis.

According to the present invention, a process for preparing a compoundlibrary in the same vessel is provided, comprising: (1) mixing one ormore donor substrates, acceptor substrates, and transferases; (2)performing a transfer reaction to reach a degree of transfer of 1%-99%by incubating the mixture; and (3) stopping the transfer reaction.

The vessel used in the process of the present invention can beappropriately selected by those skilled in the art in the steps ofpreparing a compound library. Preferably, it is a microtube, plate, orflask, more preferably a microtube or plate, most preferably amicrotube.

The donor substrate, acceptor substrate, and transferase used in theprocess of the present invention are as described in detail above. Thedonor substrate and acceptor substrate used in step (1) are not limitedin the types to be mixed so far as the transferase is substrate-specificfor them. The transferase used in the same step is not limited in thetype to be added so far as it is substrate-specific for the donorsubstrate and the acceptor substrate. For the incubation in step (2),any apparatus can be used so far as it can maintain a reaction periodand a reaction temperature suitable for the transferase to produce theactivity of transferring a substituent. The apparatus is preferably atemperature-controlled shaker, PCR system, incubator,temperature-controlled bath or heat block, more preferably atemperature-controlled shaker or PCR system. The reaction period of thetransferase means a period required to reach a predetermined degree ofreaction to which a substituent is transferred from a donor substrate toan acceptor substrate after the transferase is added, as describedbelow, and varies with the predetermined degree of reaction and reactionconditions of each transferase.

In one embodiment of the present invention, steps (1)-(3) may berepeated once or more using the same or different donor substrate andtransferase as or from the donor substrate and transferase used in step(1). A compound library containing specific proportions of a pluralityof compounds having desired structures can be obtained by appropriatelyselecting a donor substrate and a transferase.

Means for stopping the transfer reaction in step (3) is not specificallylimited so far as the transferase can be deactivated or removed when apredetermined degree of transfer is reached. Preferably, it is heattreatment, addition of a protein modifier, addition of an inhibitor,filtration or solid phase extraction, more preferably heat treatment orfiltration. Here, the degree of transfer at which the transfer reactionis stopped in step (3) can be appropriately determined by those skilledin the art to prepare a compound library containing a predeterminedamount of a specific structure. For example, a mixing ratio of 1:1between a compound having one substituent and a compound not having itis achieved by stopping the reaction when the degree of reaction by thetransferase reaches 50% during the process for preparing a compoundlibrary. On the other hand, the proportion of the presence of a specificsubstituent can be modulated by changing the degree of transfer in therange of 1-99% rather than 50%. The degree of transfer by thetransferase in the process for preparing a compound library of thepresent invention is preferably about 1-99%, more preferably about5-95%, still more preferably about 10-90%, further more preferably about20-80%, still further more preferably about 30-70%, even more preferablyabout 40-60%, most preferably about 50%. For example, when the degree oftransfer by each transferase in the first and second stages is 50% inscheme 2, the ratio of compound A, compound C, compound B and compound Dproduced by the two stages of reaction is 1:1:1:1. When the degree oftransfer in each reaction stage is 40%, a compound library containingcompound A: compound C: compound B compound D=9:6:6:4 can be obtained.Thus, compound libraries containing different ratios of componentshaving specific structures produced can be constructed by changing thedegrees of transfer by various transferases used.

In one embodiment of the present invention wherein steps (1)-(3) arerepeated, a step in which the degree of transfer is 100% may be includedin a partial transfer reaction. By partially including such a step, acompound library having an increased proportion of only a componenthaving a specific structure can be prepared.

In one embodiment of the present invention, the step of collecting apart of an unreacted acceptor substrate before starting a transferreaction and adding the collected acceptor substrate to the productafter stopping the transfer reaction may be included in order to preparea more diverse compound library. By including this reaction step, adiverse compound library can be prepared.

In one embodiment of the present invention, a mixture at a mid stage forconstructing a library can be used as a starting material forconstructing another library in the process for preparing a compoundlibrary of the present invention.

The degree of transfer in each transfer reaction can be determined bymeasuring, but not limited to, the concentration, composition ratios orweight ratios of unreacted materials and the reaction product insolution. A preferred method is mass spectrometry, in which a part ofthe reaction solution is collected and can be measured for the amount(or composition ratio) of each component present in the reactionsolution by a mass spectrometer. This determination of the degree oftransfer by mass spectrometry is based on the fact that each massspectrum (m/z) shown in the spectra after mass spectrometry correspondsto the structure of one compound. Unreacted materials, the product andamounts (or composition ratios) thereof, or the structure of eachcomponent contained in the reaction solution can be rapidly and readilyidentified by one measurement using a mass spectrometer withoutnecessity of isolating/purifying the product in each reaction step.Thus, a compound library containing components each having a knownstructure can be prepared by the process for preparing a compoundlibrary of the present invention.

The “mass spectrometry” is an analytical method for mostly measuring themass of a sample using a gas phase ion spectrometer comprising a sampleintroduction part, an ionization part and a mass spectrometric part(mass separation part and detection part). Specifically, a sample isionized in an ionization part (or apparatus), and the resulting ionizedmolecules are separated according to the mass/charge (m/z) in a massspectrometric part and detected in a detection part. As used herein, theterm “mass spectrometer” means an apparatus capable of testing a sample,e.g., biological molecules such as oligosaccharides, sugars, proteins,peptides and nucleic acids by mass spectrometry. Methods for ionizingbiological molecules include, but not limited to, Matrix-Assisted LaserDesorption Ionization (MALDI), Laser Desorption (LD), Fast AtomBombardment (FAB), Liquid Secondary Ion Mass Spectrometry (LSIMS),Liquid Ionization (LI), Electrospray Ionization (ESI), and AtmosphericPressure Chemical Ionization (APCI). Ions of ionized biologicalmolecules are separated according to the mass/charge (m/z) inherent tothe biological molecules by using electromagnetic interactions. The massspectrometric part for separating and detecting ions is an analyzerincluding, but not limited to, time-of-flight (TOF), quadrupole ion traptime-of-flight (QIT-TOF), quadrupole, ion trap, magnetic sector, andFourier transform ion cyclotron resonance (FT-ICR) analyzers. Examplesof MALDI-TOF mass spectrometers include Ettan MALDI-TOF Pro (AmershamBiosciences), and Reflex IV (Brucker).

In one embodiment of the present invention, the number of cycles ofsteps (1)-(3) can be appropriately determined by those skilled in theart to obtain a compound library having a desired structure. In order toprepare a compound library having multiple structures, the number ofcycles of steps (1)-(3) is preferably increased. The number of cycles ispreferably one or more, more preferably 1-50, still more preferably1-10, further more preferably 1-8, most preferably 1-6. For example, anoligosaccharide library containing a maximum of eight oligosaccharideswas successfully constructed via four stages of glycosylation reactionsfrom one glycosyl acceptor substrate (β4Gal-core 2-Muc1a) by repeating 4cycles of steps (1)-(3) in Example 1 below. In Example 2, anoligosaccharide library consisting of eight polylactosamines havingdifferent repetition numbers of lactosamines was successfullyconstructed by only one cycle of steps (1)-(3) from one glycosylacceptor substrate (Tn-Muc1a).

In one embodiment of the present invention, the order of transferasesused in the process for preparing a compound library of the presentinvention is not limited, but those skilled in the art can prepare acompound library containing a specific structure based on the substratespecificity of transferases by selecting the types of transferases andthe order of adding them to contain a compound having a specificstructure. For example, two oligosaccharides that were not obtained whenan N-acetylneuraminyltransferase, a fucosyltransferase, anN-acetylglucosaminyltransferase, and a galactosyltransferase were addedin this order to the reaction system were obtained by changing the orderof adding the fucosyltransferase (second) and theN-acetylglucosaminyltransferase (third), as described in Example 1below. On the other hand, a compound library not containing a specificstructure can also be constructed by changing the combination oftransferases.

4. Compound Libraries

According to the present invention, compound libraries prepared by theprocesses described above are provided. In the processes for preparing acompound library of the present invention, compound libraries containingmultiple known structures can be rapidly and easily prepared, unlikeconventional methods.

Compound libraries of the present invention contain certain proportionsof components having multiple structures depending on the types of theacceptor substrate, donor substrate and transferase used, the degree oftransfer, and the number of cycles of steps (1)-(3) as described above.The amount of a specific compound among components contained in thecompound libraries can be modulated by changing the degree of reactionin the range of 1-99% and/or changing the order of adding thetransferases used.

In one embodiment of the present invention, a compound library isprovided characterized in that the structure of each component of thecompound library is identified from its molecular weight.

More specifically, an oligosaccharide library containing a total of sixoligosaccharides, i.e.,

(i) Galβ1-4GlcNAcβ1-6(Galβ1-3)GalNAcα1-Muc1a,

(ii) Galβ1-4(Fucα1-3)GlcNAcβ1-6(Galβ1-3)GalNAcα1-Muc1a,

(iii) GlcNAcβ1-3Galβ1-4GlcNAcβ1-6(Galβ1-3)GalNAcα1-Muc1a,

(iv) Galβ1-4GlcNAcβ1-3Galβ1-4GlcNAcβ1-6(Galβ1-3)GalNAcα1-Muc1a,

(v) Neu5Acα2-3Galβ1-4GlcNAcβ1-6(Galβ1-3)GalNAcα1-Muc1a, and

(vi) Neu5Acα2-3Galβ1-4(Fucα1-3)GlcNAcβ1-6(Galβ1-3)GalNAcα1-Muc1a

was successfully constructed by using β4Gal-core 2-Muc1a(Ala-His-Gly-Val-Thr-Ser-Ala-Pro-Asp-Thr-Arg (SEQ ID NO: 1)) as aglycosyl acceptor substrate (starting material) along with anN-acetylneuraminyltransferase (ST3Gal IV), a fucosyltransferase (FUT6),an N-acetylglucosaminyltransferase (β3GnT2), and a galactosyltransferase(β4GalT1) in this order at a glycosylation degree of 50% by eachglycosyltransferase, as described in Example 1 below (see FIG. 1). Asused herein, Gal means galactose, GlcNAc means N-acetylglucosamine,GalNAc means N-acetylgalactosamine, Fuc means fucose, and Neu5Ac meansN-acetylneuraminic acid. In this process, there is a possibility thattwo other oligosaccharides (as indicated by crosses in scheme 3 below)may be theoretically synthesized in view of the types ofglycosyltransferases added, but such oligosaccharides were not obtainedby the reaction sequence above. However, the oligosaccharides that werenot obtained by the previous method, i.e.,GlcNAcβ1-3Galβ1-4(Fucα1-3)GlcNAcβ1-6(Galβ1-3)GalNAcα1-Muc1a, andGalβ1-4GlcNAcβ1-3Galβ1-4(Fucα1-3)GlcNAcβ1-6(Galβ1-3)GalNAcα1-Muc1a weresuccessfully obtained by changing the order of adding thefucosyltransferase (second) and the N-acetylglucosaminyltransferase(third). These oligosaccharide structures could be readily determined bya mass spectrometer.

As described in Example 2 below, an oligosaccharide library containingoligosaccharides elongated by mostly 4-11 units of a lactosaminestructure (Galβ1-4GlcNAcβ1-3) was successfully constructed in onereaction without repeating steps (1)-(3) when Tn-Muc1a was used as astarting material and two glycosyl donor substrates (UDP-GlcNAc andUDP-Gal) and three glycosyltransferases (twoN-acetylglucosaminyltransferases and a galactosyltransferase) were added(see FIG. 3). Moreover, an oligosaccharide having an N-acetylneuraminicacid residue at the oligosaccharide elongation end was also successfullyprepared by using an N-acetylneuraminyltransferase and CMP-Neu5Ac onthis lactosamine structure.

On the other hand, an oligosaccharide library containing sialyl Lewis A(Neu5Acα2-3Galβ1-3(Fucα1-4)GlcNAcβ1-3GalNAcα1-), sialyl Lewis X(Neu5Acα2-3Galβ1-4(Fucα1-3)GlcNAcβ1-3GalNAcα1-), antigen A(GalNAcα1-3(Fucα1-2)Galβ1-3GlcNAcβ1-3GalNAcα1-), or antigen B(Galα1-3(Fucα1-2)Galβ1-3GlcNAcβ1-3GalNAcα1-) can be obtained byrepeating 2-3 cycles of steps (1)-(3) using core 3 (GlcNAcβ1-3GalNAcα1-)structure as a starting material with various glycosyltransferases andglycosyl donor substrates, as described in Example 4 below. Morespecifically, when an oligosaccharide library containing sialyl Lewis Ais to be prepared, core 3 structure is mixed with galactose as aglycosyl donor substrate to give Galβ1-3 core 3 structure via agalactosyltransferase-mediated transfer reaction and the product isfurther reacted with N-acetylneuraminic acid and fucose as glycosyldonor substrates in the presence of an N-acetylneuraminyltransferase anda fucosyltransferase, whereby an oligosaccharide library containingsialyl Lewis A and other three oligosaccharides(Galβ1-3GlcNAcβ1-3GalNAcα1-, Galβ1-3(Fucα1-4)GlcNAcβ1-3GalNAcα1-,Neu5Acα2-3Galβ1-3GlcNAcβ1-3GalNAcα1-) can be obtained.

5. Chips

According to the present invention, chips suitable for applying compoundlibraries of the present invention for diagnoses of diseases or otherpurposes are provided. As used herein, the term “chip” means a compoundlibrary of the present invention on a solid substrate, e.g., a chip,membrane, filter or glass. Specifically, by using a chip of the presentinvention, molecules or the like that react with a specific compound ina compound library on the chip can be rapidly detected. For example,when an oligosaccharide library is used on a chip,oligosaccharide-recognizing molecules, e.g., lectins, viruses, microbes,toxins, and bacteria recognize an oligosaccharide immobilized on thechip and bind or otherwise react with it, whereby a signal from eacholigosaccharide is detected and the data obtained are analyzed. Methodsfor immobilizing compounds in compound libraries on solid substratesinclude, but not limited to, hydrophobic bonds to substrate surfaces, oramide bonds or sulfide bonds to chemically modified plate surfaces.

The following examples further illustrate the present invention with nointention to limit the technical scope of the present invention thereto.Those skilled in the art can easily add modifications/changes to thepresent invention on the basis of the description herein, and thesemodifications/changes are also included in the technical scope of thepresent invention.

Example 1 Construction of Oligosaccharide Libraries from β4Gal-Core2-Muc1a

A solution containing a sialyltransferase (ST3Gal IV), a glycosylacceptor substrate (β4Gal-core 2-Muc1a), a glycosyl donor substrate(CMP-Neu5Ac), a divalent cation, a buffer and others in the volumesshown in Table 2A below was incubated at 37° C. for 20 hours. Thereaction process was monitored on a mixture of a 0.1 μL sample of thisreaction solution and 0.5 μL of 2,5-dihydrobenzoic acid (DHB) using amass spectrometer (Reflex IV (Brucker)). At the point when the reactionproceeded to about 50%, the reaction was stopped by heating the reactionsolution at 100° C. for 5 minutes to deactivate the enzyme. Thisreaction solution is designated “reaction solution 1” (reaction 1).Then, a fucosyltransferase (FUT6) and a glycosyl donor substrate(GDP-Fuc) in the volumes shown in Table 2B were added to reactionsolution 1, and the mixture was reacted at 25° C. for 30 minutes. Thisenzymatic reaction solution was monitored by Reflex IV, and afterconfirming that the reaction has proceeded to about 50%, the enzyme wasdeactivated by heating at 100° C. for 5 minutes. This reaction solutionis designated “reaction solution 2” (reaction 2). Further, anN-acetylglucosaminyltransferase (β3GnT2) and a glycosyl donor substrate(UDP-GlcNAc) in the volumes shown in Table 2C were added to reactionsolution 2, and the mixture was reacted at 37° C. for 2 hours. Afterconfirming that the reaction has proceeded to about 50% as monitored byReflex IV, the reaction was stopped by heating at 100° C. for 5 minutes.This reaction solution is designated “reaction solution 3” (reaction 3).Then, a galactosyltransferase (β4GalT1) and a glycosyl donor substrate(UDP-Gal) in the volumes shown in Table 2D were added to reactionsolution 3, and the mixture was reacted at 25° C. for 1 hour while thereaction was monitored by Reflex IV. When the reaction proceeded toabout 50%, the reaction was stopped by heating at 100° C. for 5 minutes.This reaction solution is designated “reaction solution 4” (reaction 4).ST3Gal IV 5 Acceptor (100 μM) 10 HEPES (500 mM, pH 7.0) 4 MnCl₂ (200 mM)2 CMP-Neu5Ac 1 H₂O 18 Total 40 μl FUT6 2 Reaction Solution 1 40 HEPES(500 mM, pH 7.0) 0 MnCl₂ (200 mM) 0 GDP-Fuc (1 mM) 1 Total 43 μl β3GnT21 Reaction Solution 2 43 HEPES (500 mM, pH 7.0) 0 MnCl₂ (200 mM) 0UDP-GlcNAc (1 mM) 1 Total 45 μl β4GalT1 1 Reaction Solution 3 45 HEPES(500 mM, pH 7.0) 0 MnCl₂ (200 mM) 0 UDP-GlcNAc (500 μM) 1 Total 47 μl

The results of reaction solutions 1-4 after stopping each reaction asmonitored by Reflex IV are shown in FIG. 1. In the figure, solid circlesrepresent N-acetylgalactosamine, open circles represent galactose, solidsquares represent N-acetylglucosamine, open triangles represent fucose,and open stars represent N-acetylneuraminic acid. After completion ofreaction 4, the enzyme bound to the agarose gel contained in thereaction solution was removed by filtration to prepare anoligosaccharide library containing six compounds.

When various glycosyltransferases were used in the order of reactions1-4, two oligosaccharides as shown in scheme 3 (indicated by crosses),i.e., GlcNAcβ1-3Galβ1-4(Fucα1-3)GlcNAcβ1-6(Galβ1-3)GalNAcα1-, andGalβ1-4GlcNAcβ1-3Galβ1-4(Fucα1-3)GlcNAcβ1-6(Galβ1-3)GalNAcα1- were notobtained (FIG. 1).

This result suggests that the transfer of N-acetylglucosamine by theN-acetylglucosaminyltransferase (reaction 3) was blocked by the presenceof the fucose residue attached to the N-acetylglucosamine residue. Thus,an oligosaccharide library not having a specific oligosaccharidestructure could be constructed by the combination and the order ofaddition of enzymes described above.

For the purpose of obtaining an oligosaccharide library containing thetwo oligosaccharides that were not obtained by the order of reactionsdescribed above, similar transfer reactions were performed with theorder of addition of glycosyltransferases changed. A solution containinga sialyltransferase (ST3Gal IV), a glycosyl acceptor substrate(β4Gal-core 2-Muc1a), a glycosyl donor substrate (CMP-Neu5Ac), adivalent cation, a buffer and others in the volumes shown in Table 3Abelow was reacted at 37° C. for 20 hours while the reaction wasmonitored by Reflex IV in the same manner as described above, and at thepoint when the reaction proceeded to about 50%, the reaction was stoppedby heat treatment. This reaction solution is designated “reactionsolution 1′” (reaction 1′). Then, an N-acetylglucosaminyltransferase(β3GnT2) and a glycosyl donor substrate (UDP-GlcNAc) in the volumesshown in Table 3B were added, and the mixture was reacted at 37° C. for2 hours, and at the point when the reaction proceeded to about 50%, thereaction was stopped in the same manner. This reaction solution isdesignated “reaction solution 2′” (reaction 2′). Further, afucosyltransferase (FUT6) and a glycosyl donor substrate (GDP-Fuc) inthe volumes shown in Table 3C were added, and the mixture was reacted at25° C. for 30 minutes, after which the reaction was stopped by heattreatment. This reaction solution is designated “reaction solution 3′”(reaction 3′). Then, a galactosyltransferase (β4GalT1) and a glycosyldonor substrate (UDP-Gal) in the volumes shown in Table 3D were added,and the mixture was reacted at 25° C. for 2 hours, after which thereaction was stopped by heat treatment. This reaction solution isdesignated “reaction solution 4” (reaction 4′). ST3Gal IV 5 Acceptor(100 μM) 10 HEPES (500 mM, pH 7.0) 4 MnCl₂ (200 mM) 2 CMP-Neu5Ac (50 mM)2 H₂O 18 Total 40 μl β3GnT2 2 Reaction Solution 1 40 HEPES (500 mM, pH7.0) 0 MnCl₂ (200 mM) 0 UDP-GlcNAc (1 mM) 1 Total 43 μl FUT6 1 ReactionSoution 2 43 HEPES (500 mM, pH 7.0) 0 MnCl₂ (200 mM) 0 GDP-Fuc (1 mM) 1Total 45 μl β4GalT1 1 Reaction Solution 3 45 HEPES (500 mM, pH 7.0) 0MnCl₂ (200 mM) 0 UDP-Gal (500 μM) 1 Total 47 μl

The results of reaction solutions 1′-4′ after stopping each reaction asmonitored by Reflex IV are shown in FIG. 2. As shown in FIG. 2 andscheme 1, an oligosaccharide library containing two oligosaccharidestructures that were not obtained by reactions 1-4 could be obtained byreactions 1′-4′.

These results show that oligosaccharide libraries having a desiredoligosaccharide structure can be constructed by changing the order ofglycosyltransferases added. On the other hand, oligosaccharide librariescan also be constructed not to contain a specific oligosaccharidestructure.

Example 2 Construction of Oligosaccharide Libraries of Polylactosamines

Elongation of polylactosamine chains was performed using Tn-Muc1a as astarting material in the same vessel. N-acetylglucosaminyltransferases(β3GnT6 and β3GnT2), a galactosyltransferase (β4GalT1), a glycosylacceptor substrate (Tn-Muc1a), and glycosyl donor substrates (UDP-GlcNAcand UDP-Gal) in the volumes shown in Table 4 below were used. β3GnT6 isan enzyme transferring N-acetylglucosamine to the GalNAc residue of Tnantigen via β1-3 linkage, and β3GnT2 is an enzyme transferringN-acetylglucosamine to the galactose residue of lactosamine chains viaβ1-3 linkage. A solution containing these components as well as adivalent cation, a buffer and others was reacted at 37° C. for 30 hourswhile the reaction was monitored by Reflex IV in the same manner as inExamples 1 and 2 to show that a mixture containing oligosaccharidestructures consisting of Galβ1-4-core 3 structures elongated by mostly4-11 units of a lactosamine structure (Galβ1-4GlcNAcβ1-3) wassuccessfully obtained in one reaction as monitored by MALDI TOF MS (FIG.3). β3GnT6 10 β3GnT2 5 β4GalT1 5 Tn-Muclα (500 μM) 5 HEPES (500 mM, pH7.0) 6 MnCl₂ (200 mM) 3 UDP-GlcNAc (50 mM) 5 UDP-Gal (50 mM) 5 H₂O 16Total 60 μl

Example 3 Construction of Oligosaccharide Libraries of SialylatedPolylactosamines

A solution containing an N-acetylglucosaminyltransferase (β3GnT2), agalactosyltransferase (β4GalT1), a glycosyl acceptor substrate (core3-Muc1a), glycosyl donor substrates (UDP-GlcNAc and UDP-Gal), a divalentcation, a buffer and others in the volumes shown in Table 5A below wasreacted at 37° C. for 12 hours. The reaction was monitored by Reflex IVto show that an oligosaccharide library containing oligosaccharidestructures having core 3 structure elongated by mostly 1-5 units of alactosamine structure could be obtained in one reaction. Then, thereaction was stopped by heat treatment. This reaction solution isdesignated “reaction solution A” (reaction A).

Further, a sialyltransferase (ST3Gal III) and a glycosyl donor substrate(CMP-Neu5Ac) in the volumes shown in Table 5B were added to reactionsolution A obtained as above, and the mixture was reacted at 37° C. for20 hours, and the reaction was stopped by heat reaction (reaction B).The results monitored by Reflex IV are shown in FIG. 4. Thus, anoligosaccharide library of polylactosamine chains sialylated at thereducing end was successfully constructed. β3GnT2 5 β4GalT1 5Core3-Muclα (500 μM) 5 HEPES (500 mM, pH 7.0) 2 MnCl2 (200 mM) 2UDP-GlcNAc (10 mM) 5 UDP-Gal (10 mM) 5 H₂O 11 Total 40 μl ST3Gal III 5Reaction Solution A 40 HEPES (500 mM, pH 7.0) 0 MnCl2 (200 mM) 0CMP-Neu5Ac (50 mM) 2 H₂O 0 Total 60 μl

Example 4 Construction of Various Oligosaccharide Libraries Having Core3 Structure

By using core 3 structure as a starting material along with combinationsof various glycosyltransferases, various oligosaccharide librariescontaining sialyl Lewis A, sialyl Lewis X, antigen A, or antigen B canbe constructed as shown in scheme 4 below.

(1) An Oligosaccharide Library Containing Sialyl Lewis A

A galactosyltransferase transferring galactose to theN-acetylglucosamine residue via β1-3 linkage (β3GalT5), a glycosylacceptor substrate (core 3: GlcNAcβ1-3GalNAcα1-), and a glycosyl donorsubstrate (UDP-Gal) are reacted for a predetermined period, and thereaction is stopped when the degree of reaction reaches about 100%(reaction 1). This reaction affords Galβ1-3GlcNAcβ1-3GalNAcα1-. Thisreaction solution is designated “reaction solution a”. Then, asialyltransferase (ST3Gal I), a fucosyltransferase (FUT3) and glycosyldonor substrates (CMP-Neu5Ac and GDP-Fuc) are added to reaction solutiona, and the mixture is reacted for a predetermined period, whereby anoligosaccharide library having four oligosaccharide structuresconsisting of sialyl Lewis A(Neu5Acα2-3Galβ1-3(Fucα1-4)GlcNAcβ1-3GalNAcα1-) and other threeoligosaccharides (Galβ1-3GlcNAcβ1-3GalNAcα1-,Galβ1-3(Fucα1-4)GlcNAcβ1-3GalNAcα1-,Neu5Acα2-3Galβ1-3GlcNAcβ1-3GalNAcα1-) can be constructed (scheme 4).

(2) An Oligosaccharide Library Containing Sialyl Lewis X

A galactosyltransferase transferring galactose to theN-acetylglucosamine residue via β1-4 linkage (β4GalT1), a glycosylacceptor substrate (core 3: GlcNAcβ1-3GalNAcα1-), and a glycosyl donorsubstrate (UDP-Gal) are reacted for a predetermined period, and thereaction is stopped when the degree of reaction reaches about 100%. Thisreaction affords Galβ1-4GlcNAcβ1-3GalNAcα1-. This reaction solution isdesignated “reaction solution b”. Then, a sialyltransferase (ST3GalIII), an N-acetylglucosaminyltransferase (β3GnT2), a fucosyltransferase(FUT6), a galactosyltransferase (β4GalT1), and glycosyl donor substrates(CMP-Neu5Ac, UDP-GlcNAc, GDP-Fuc, UDP-Gal) are added to reactionsolution b, and the mixture is reacted for a predetermined period,whereby an oligosaccharide library having eight oligosaccharidestructures consisting of sialyl Lewis X(Neu5Acα2-3Galβ1-4(Fucα1-3)GlcNAcβ1-3GalNAcα1-) and other sevenoligosaccharides can be constructed (scheme 4).

(3) An Oligosaccharide Library Containing Antigen A

Reaction solution a prepared in (1) above is reacted with asialyltransferase (ST6GalNAc I), a fucosyltransferase (FUT2), anN-acetylgalactosaminyltransferase (H-α3GalNAcT), and glycosyl donorsubstrates (CMP-Neu5Ac, GDP-Fuc, UDP-GalNAc) for a predetermined period,and the reaction is stopped when the degree of reaction reaches about50%. As a result, an oligosaccharide library containing a total of sixoligosaccharides including antigen A(GalNAcα1-3(Fucα1-2)Galβ1-3GlcNAcβ1-3GalNAcα1-) can be constructed(scheme 4).

(4) An Oligosaccharide Library Containing Antigen B

Reaction solution a prepared in (1) above is reacted with asialyltransferase (ST6GalNAc I), a fucosyltransferase (FUT2), agalactosyltransferase (H-α3GalT), and glycosyl donor substrates(CMP-Neu5Ac, GDP-Fuc, UDP-Gal) for a predetermined period and thereaction is stopped when the degree of reaction reaches about 50%. As aresult, an oligosaccharide library containing a total of sixoligosaccharides including antigen B(Galα1-3(Fucα1-2)Galβ1-3GlcNAcβ1-3GalNAcα1-) can be constructed (scheme4).

INDUSTRIAL APPLICABILITY

Oligosaccharide libraries are established, which can be applied todiagnoses and treatments of various diseases by analyzing interactionsbetween oligosaccharides and oligosaccharide-related molecules andcorrelations between oligosaccharide structures and their functions.

REFERENCES

A. Patent Documents

1. International Publication WO03/057887

B. Non-Patent Documents

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1. A process for preparing a compound library in the same vessel,comprising: (1) mixing one or more donor substrates, acceptorsubstrates, and transferases; (2) performing a transfer reaction toreach a degree of transfer of 1%-99% by incubating the mixture; and (3)stopping the transfer reaction.
 2. The process for preparing a compoundlibrary of claim 1 wherein the degree of transfer is measured by a massspectrometer.
 3. The process for preparing a compound library of claim 1wherein step (1) comprises adding the donor substrate and thetransferase to the acceptor substrate.
 4. The process for preparing acompound library of claim 3, further comprising the step of adding thesame or different donor substrate and transferase to the product of thetransfer reaction and repeating one or more cycles of steps (1)-(3). 5.The process for preparing a compound library of claim 4 wherein thenumber of cycles of steps (1)-(3) is 1-10.
 6. The process for preparinga compound library of claim 5 wherein the reaction is stopped when thedegree of transfer reaches 50%.
 7. The process for preparing a compoundlibrary of claim 1 wherein the transferase is anN-acetylneuraminyltransferase, fucosyltransferase,N-acetylglucosaminyltransferase, N-acetylgalactosaminyltransferase,galactosyltransferase, glucosyltransferase, glucuronyltransferase,mannosyltransferase, xylosyltransferase, sulfotransferase,phosphotransferase, or acyltransferase.
 8. The process for preparing acompound library of claim 1 wherein the acceptor substrate is a glycosylacceptor substrate, peptide, protein or lipid or a modified form thereofor a mixture thereof.
 9. The process for preparing a compound library ofclaim 8 wherein the glycosyl acceptor substrate is a monosaccharide,oligosaccharide, glycopeptide, glycoprotein, or glycolipid.
 10. Theprocess for preparing a compound library of claim 9 wherein theoligosaccharide, glycopeptide, glycoprotein, or glycolipid has anoligosaccharide structure selected from the group consisting of Tnantigen, core 2 structure, core 3 structure, core 4 structure, core 5structure, core 7 structure, and core 8 structure.
 11. The process forpreparing a compound library of claim 1 wherein the donor substrate isselected from the group consisting of sugar nucleotides, dolicholphosphate-sugars, 3′-phosphoadenosine 5′-phosphosulfate (PAPS),adenosine triphosphate (ATP), and acetyl-CoA.
 12. A compound libraryprepared by the process of claim
 1. 13. The compound library of claim 12wherein the structure of each component of the compound library isidentified from its molecular weight.
 14. A chip using the compoundlibrary of claim 12.